Xin ZHENG， Tian-Qi ZHOU， Tao WAN， Anabel PERDICES， Jin-Quan YANG， Xin-Sheng TANG，Zheng-Ping WANG， Li-Qun HUANG， Song HUANG，3，*， Shun-Ping HE

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Huangshan population of Chinese Zacco platypus （Teleostei， Cyprinidae） harbors diverse matrilines and high genetic diversity

Xin ZHENG1，2，#， Tian-Qi ZHOU1，2，#， Tao WAN3，#， Anabel PERDICES4， Jin-Quan YANG1， Xin-Sheng TANG2，Zheng-Ping WANG5， Li-Qun HUANG6， Song HUANG2，3，*， Shun-Ping HE7，*

1Key Laboratory of Exploration and Utilization of Aquatic Genetic Resources， Shanghai Ocean University， Ministry of Education， Shanghai 201306， China
2College of Life and Environment Sciences， Huangshan University， Huangshan Anhui 245041， China
3State Key Laboratory of Genetic Resources and Evolution， Kunming Institute of Zoology， Chinese Academy of Sciences， Kunming Yunnan 650223， China
4Museo Nacional de Ciencias Naturales， CSIC， C/ José Gutiérrez Abascal， 2， 28006 Madrid， Spain
5College of Foreign Languages， Huangshan University， Huangshan Anhui 245041， China
6Landscaping Bureau of Huangshan Scenic Area， Huangshan Anhui 245000， China
7Institute of Hydrobiology， Chinese Academy of Sciences， Wuhan Hubei 430072， China

ABSTRACT

Six main mitochondrial DNA （mtDNA） lineages have been described in minnow （Zacco platypus） samples obtained from northern， western and southern China. Perdices et al. （2004） predicted that further sampling of other tributaries might discover more lineages of this species. In this study， we collected 26 Zacco platypus individuals in the Huangshan area of eastern China and determined the cytochrome b （cytb） sequence variations. Combined with reported data in GenBank， we identified ten matrilines （Zacco A-J） in a total of 169 samples， with relatively high molecular divergence found among them. The Huangshan population had the greatest genetic variation among all sampled regions and hosted six of the ten matrilines. Our results highlight the significance of the Huangshan area for the conservation of Zacco platypus.

Zacco platypus； Matriline； Huangshan；Phylogenetics； Diversity

INTRODUCTION

Zacco platypus is a common minnow that occurs in sympatry with most Chinese cyprinids （Deng et al.， 2013）. Topographical barriers may restrict its life history and drive cryptic diversity. The species’ distribution encompasses all major river systems in mainland China， as well as the Korean Peninsula and Japan（Chen， 1998）. Perdices et al. （2004） analyzed the genetic diversity of Z. platypus sampled in the upper and middle Changjiang （Yangtze River） and found four major matrilines that may harbor multiple species. Long-term interruption of dispersal is thought to have driven this diversity. Perdices and Coelho （2006） further studied samples from the Pearl River and northern drainages， and obtained six matrilines in China. Using nuclear DNA data， Berrebi et al. （2005） identified four genetic groups within Z. platypus from Sichuan， Hunan and Guangxi provinces in China.1

Although Perdices et al. （2004） predicted that exhaustive sampling of other tributaries might discover other lineages of Zacco， few specimens have been sampled in eastern China. The Huangshan area in eastern China is a mosaic of mountains with elevations lower than 2 000 m， and exhibits a complex geological history that includes tectonic movements， orogenesis，and periodic climatic change （e.g.， Ju et al.， 2007； Rüber et al.，2004； Zhang et al.， 1990）. Based on patterns of intraspecific genetic variation and buffer-zone models， Huangshan hosts refugia of eastern Asian conifers， frogs， non-migratory birds and Asian salamanders （Gao et al.， 2007； Li et al.， 2009； Murphy et al.， 2000； Wu et al.， 2013； Zhang et al.， 2008）.In view of modern genetics， genetic diversity in a given species is closely related to its adaptability， variability， and evolutionary potentiality， with genetic variation considered a prerequisite for organisms to cope with environmental uncertainty （Conrad， 1983）. Herein， we report on the genetic diversity of Z. platypus from eastern China based on extensive sampling of the Huangshan area together with prior cytb sequence data from mainland China， Taiwan （Perdices et al.， 2004； Perdices & Coelho， 2006； Wang et al.， 2007）， and Japan （He et al.， 2004； Kawamura et al.， 2014； Kitamura et al.，2012； Sasaki et al.， 2007； Wang et al.， 2007）. We further evaluated the matrilineal diversity of Z. platypus and revealed the possible ecological significance of the Huangshan area.

MATERIALS AND METHODS

Sampling

We evaluated 169 sequences in total， including 26 from five Huangshan counties （Table 1）， 137 from Perdices et al. （2004）and Perdices & Coelho （2006）， one from Wang et al. （2007），and five from Japan （AF309085， He et al. （2004）； AB198972，Sasaki et al. （2007）； AY958194， Wang et al. （2007）； AB620130，Kitamura et al. （2012）； and AB366543， Kawamura et al. （2014））. Our new samples were preserved and deposited in the Museum of Huangshan University （Voucher numbers: HUM201201-26）. Sampling sites in this study are shown in Figure 1.

Figure 1 Sampling localities （A） and main drainages of the Huangshan area （B）

PCR amplification and sequencing

Fresh dorsal muscle tissues were removed from the 26 Huangshan individuals and immediately preserved in 95% ethanol for sequencing complete mitochondrial cytb. Total DNA was extracted from tissues using standard phenol/chloroform techniques （Sambrook et al.， 1989）. Cyt b was amplified using polymerase chain reaction （PCR） with the following sets of primers: LCB1 （5'-AATGACTTGAAGAACCACCGT-3'） and HA （5'-CAACGATCTCCGGTTTACAAGAC-3'） （Brito et al.， 1997；Schmidt & Gold， 1993）. Reagents included 100 ng of template DNA， 1 μL of each primer， 5 μL of 10× reaction buffer， 2 μL dNTPs （each 2.5 mmol/L）， and 2.0 U of Taq DNA polymerase. The reactions were cycled as follows: an initial preheating at 94 °C for 3 min， 30 cycles of denaturation at 94 °C for 1 min，annealing at 55 °C for 40 s， extension at 72 °C for 1 min， and a final extension at 72 °C for 5 min. We next obtained nucleotide sequences through fractionation， purification， and sequencing according to Tiangen’s protocols. Newly obtained haplotype sequences were deposited in GenBank under Accession Nos. KM491716-35 （Table 1）.

Table 1 lnformation for samples newly obtained in this study， including localities， rivers， sample sizes， haplotypes， coordinates， voucher specimens and GenBank accession numbers

Matrilineal genealogy and population structure

We used 916 bp out of 1 140 bp of the cytb sequences in the following analyses. The newly obtained sequences and those downloaded from GenBank were aligned using Clustal X （Thompson et al.， 1997）. For phylogenetic reconstruction， two closely related species， Zacco temmincki and Candidia barbatus （Mayden et al.， 2007； Wang et al.， 2011）， were chosen as outgroups.

Bayesian inference （BI） and maximum likelihood （ML） were used to reconstruct a bifurcating tree using MrBayes v3.0 （Huelsenbeck & Ronquist， 2001） and RAxML at the CIPRES Science gateway （http: //www.phylo.org/portal2/login！input.action），respectively. JModelTest v. 0.1.1 （Posada， 2008） was used to find the best model of nucleotide evolution for ML based on the Akaike Information Criterion （AIC） and for BI based on the Bayesian Information Criterion （BIC）. Analyses selected the TN93+G model. Bayesian posterior probabilities （BPP） and the frequencies of nodal resolution were obtained by Markov Chain Monte Carlo （MCMC） analysis with one cold chain and three heated chains. The BI analysis used 10 000 000 generations，with sampling every 1 000 generations and discarding the first 3x106generations as burn-in. We ran four analyses starting with random trees and a consensus of the resulting 36 000 trees was computed from all four runs.

Estimation of divergence time

Divergence times among the main lineages of Z. platypus were estimated using a Bayesian MCMC approach implemented in BEAST V.1.7.5 based on a strict molecular clock （Drummond & Rambaut， 2007）. The parameters were: substitution model，TN93+G； tree prior， Coalescent: constant size； normal distribution； 10 million generations； parameters logged every 1 000； burn-in value=1 000. The molecular clock of cyprinids was assumed to be 1.52% site-1Ma （million years）-1（Doadrio et al.，2002） for cyt b.

RESULTS

A total of 75 haplotypes were defined from all 169 in-group individuals. The topologies of the BI and ML trees were nearly identical （Figure 2）. The haplotypes were grouped into main clade 1 and 2. Clade 1 hosted individuals from Huangshan， Sichuan，Hunan and Guangxi and clade 2 contained specimens from Huangshan， Beijing and Japan. We identified ten matrilines of Z. platypus according to the topology of the phylogenetic tree and the genetic variation between the ten matrilines.

Figure 2 Phylogenetic tree derived from the maximum likelihood of the cyt b sequences

Six of the ten lineages involved Huangshan individuals， and four consisted entirely of Huangshan individuals. Moreover，samples from the same Huangshan location were grouped into different clades. For example， Qimen had samples from matrilines A， F and J， and Xiuning had samples from matrilines A and I. The genetic divergences of these samples were significant， and the maximum pairwise differences from the same counties reached 6.0% （Xiuning） and 21.9% （Qimen）.

We grouped sampling localities into seven geographic units according to geographic distances and then calculated the nucleotide diversity within them. Huangshan showed remarkably high nucleotide diversity relative to other groups （2.5-111.7 times that of others） although the geographical area of Huangshan was less than that of the other units （Table 3）.

Table 2 Matrix of pairwise genetic variation between matrilines （A-J） of Z. platypus

Table 3 Matrilines， haplotype （h）， and nucleotide diversity （π） with standard errors （SE） for each geographic unit

Divergence times estimated for the in-group nodes are shown in Figure 3. The initial divergence occurred at about 10.67 Ma.

Figure 3 Time tree of Z. platypus

DISCUSSION

Perdices et al. （2004） and Perdices & Coelho （2006） divided Z. platypus sampled in southern， western and northern China into matrilines A-F. They suggested that the long-term interruptionof gene flow might have caused the diversification and an underestimation of the number of species. Our analyses identified ten matrilines of Z. platypus in Chinese and some Japanese populations. This confirms the prediction of Perdices et al. （2004， page: 9） that “exhaustive sampling of other tributaries might evidence other Zacco lineages”. This is also in accordance with that found for Opsariichthys bidens， a sympatric species of Z. platypus （Perdices et al.， 2005）.

Some drainages still await sampling， such as the Yellow River， one of the most important drainages in China. Future research should detect additional matrilines of Zacco， while morphological analyses may help differentiate morphological differences of taxonomic significance.

Grant & Bowen （1998） interpreted four basic population history scenarios based on haplotype and nucleotide diversities，which can also be used to clarify the history of Z. platypus populations. Our results revealed a pattern of high haplotype and nucleotide diversity in the Huangshan population （Table 3），which likely indicates large stable populations with long evolutionary histories or secondary contact between differentiated lineages （Grant & Bowen， 1998）. The highest levels of genetic variation may occur in the region of origin. For example， Savolainen et al. （2002） claimed an East Asian origin for the domestic dog in part due to the area having the highest level of genetic diversity.

Genetic variability in mtDNA has been reported in fish species. Several scenarios have been proposed to explain the maintenance of high haplotype diversity within populations，including large population size， environmental heterogeneity，and life history traits that favor rapid population increase （Han et al.， 2008； Ju et al.， 2013； Yang et al.， 2012）. Huangshan has a heterogeneous topography， with mountains of elevation lower than 2 000 m maintaining stable climatic conditions during the Pleistocene. This condition likely provided glacial refugia for many species （Gao et al.， 2007； Li et al.， 2009； Qian & Ricklefs， 2000；Wu et al.， 2013； Zhang et al.， 2008）. At least three other species or species groups have high levels of nucleotide diversity in the Huangshan area， including the Chinese giant salamander， sharpsnouted pit viper and Asian salamander （Huang et al.， 2007；Murphy et al.， 2000； Wu et al.， 2013）. These co-occurrences indicate that Huangshan hosts old lineages.

We only used mtDNA for genetic analyses. Therefore， it will be necessary to gather and analyze nuclear DNA data in the future to assess population structure and gene flow and thus better inform the demographic history of this fish species.

ACKNOWLEDGEMENTS

We extend our thanks to Dr. Robert W. MURPHY for his valuable comments on this manuscript. We thank Jin-Min CHEN （Yunnan University）， Bao-Lin ZHANG （Kunming Institute of Zoology， CAS） and Li-Fang PENG （Nanjing Forestry University） for their help in data processing. We further thank Dian-Cheng YANG （Nanjing Forestry University） and Jun-Sheng CUI （Anhui Agricultural University） for sampling.

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10.13918/j.issn.2095-8137.2016.2.103

18 June 2015； Accepted: 25 February 2016

Foundation items: This research was funded by the National Natural Science Foundation of China （NSFC 30870290， 31071891 and 31471968）

*Corresponding authors， E-mail: snakeman@hsu.edu.cn；clad@ihb.ac.cn

#Authors contributed equally to this work